Iron uptake and transfer from ceruloplasmin to transferrin

Background

Dietary and recycled iron are in the Fe^2 + oxidation state. However, the metal is transported in serum by transferrin as Fe^3 +. The multi-copper ferroxidase ceruloplasmin is suspected to be the missing link between acquired Fe^2 + and transported Fe^3 +.

Methods

This study uses the techniques of chemical relaxation and spectrophotometric detection.

Results

Under anaerobic conditions, ceruloplasmin captures and oxidizes two Fe^2 +. The first uptake occurs in domain 6 (< 1 ms) at the divalent iron-binding site. It is accompanied by Fe^2 + oxidation by Cu^2 +_D6. Fe^3 + is then transferred from the binding site to the holding site. Cu⁺_D6 is then re-oxidized by a Cu^2 + of the trinuclear cluster in about 200 ms. The second Fe^2 + uptake and oxidation involve domain 4 and are under the kinetic control of a 200 s change in the protein conformation. With transferrin and in the formed ceruloplasmin–transferrin adduct, two Fe^3 + are transferred from their holding sites to two C-lobes of two transferrins. The first transfer (~ 100 s) is followed by conformation changes (500 s) leading to the release of monoferric transferrin. The second transfer occurs in two steps in the 1000–10,000 second range.

Conclusion

Fe^3 + is transferred after Fe^2 + uptake and oxidation by ceruloplasmin to the C-lobe of transferrin in a protein–protein adduct. This adduct is in a permanent state of equilibrium with all the metal-free or bounded ceruloplasmin and transferrin species present in the medium.

General significance

Ceruloplasmin is a go-between dietary or recycled Fe^2 + and transferrin transported Fe^3 +.     

Transferrin receptor-1 iron-acquisition pathway — Synthesis,kinetics, thermodynamics and rapid cellular internalization of a holotransferrin–maghemite nanoparticle construct

Background

Targeting nanoobjects via the iron-acquisition pathway is always reported slower than the transferrin/receptor endocytosis. Is there a remedy?

Methods

Maghemite superparamagnetic and theragnostic nanoparticles (diameter 8.6 nm) were synthesized, coated with 3-aminopropyltriethoxysilane (NP) and coupled to four holotransferrin (TFe₂) by amide bonds (TFe₂–NP). The constructs were characterized by X-ray diffraction, transmission electron microscopy, FTIR, X-ray Electron Spectroscopy, Inductively Coupled Plasma with Atomic Emission Spectrometry. The in-vitro protein/protein interaction of TFe₂–NP with transferrin receptor-1 (R1) and endocytosis in HeLa cells were investigated spectrophotometrically, by fast T-jump kinetics and confocal microscopy.

Results

In-vitro, R1 interacts with TFe₂–NP with an overall dissociation constant K_D = 11 nM. This interaction occurs in two steps: in the first, the C-lobe of the TFe₂–NP interacts with R1 in 50 μs: second-order rate constant, k₁ = 6 × 10¹⁰ M^− 1 s^− 1; first-order rate constant, k_− 1 = 9 × 10⁴ s^− 1; dissociation constant, K_1d = 1.5 μM. In the second step, the protein/protein adduct undergoes a slow (10,000 s) change in conformation to reach equilibrium. This mechanism is identical to that occurring with the free TFe₂. In HeLa cells, TFe₂–NP is internalized in the cytosol in less than 15 min.

Conclusion

This is the first time that a nanoparticle–transferrin construct is shown to interact with R1 and is internalized in time scales similar to those of the free holotransferrin.

General significance

TFe₂–NP behaves as free TFe₂ and constitutes a model for rapidly targeting theragnostic devices via the main iron-acquisition pathway.     

Heme binding to human alpha-1 proteinase inhibitor

Background

Heme is a unique prosthetic group of various hemoproteins that perform diverse biological functions; however, in its free form heme is intrinsically toxic in vivo. Due to its potential toxicity, heme binding to plasma proteins is an important safety issue in regard to protein therapeutics derived from human blood. While heme binding by hemopexin, albumin and α₁-microglobulin has been extensively studied, the role of other plasma proteins remains largely unknown.

Methods

We examined two acute-phase plasma proteins, haptoglobin (Hp) and alpha-1 proteinase inhibitor (α₁-PI) for possible interactions with heme and bilirubin (BR), the final product of heme degradation, using various techniques: UV/Vis spectroscopy, fluorescence, circular dichroism (CD), and surface plasmon resonance (SPR).

Results

According to our data, Hp exhibits a very weak association with both heme and BR; α₁-PI's affinity to BR is also very low. However, α₁-PI's affinity to heme (K_D 2.0 × 10^− 8 M) is of the same order of magnitude as that of albumin (1.26 × 10^− 8 M). The data for α₁-PI binding with protoporphyrin IX (PPIX) suggest that the elimination of the iron atom from the porphyrin structure results in almost 350-fold lower affinity (K_D 6.93 × 10^− 6 M), thus indicating that iron is essential for the heme coordination with the α₁-PI.

Conclusions

This work demonstrates for the first time that human α₁-PI is a heme binding protein with an affinity to heme comparable to that of albumin.

General significance

Our data may have important implications for safety and efficacy of plasma protein therapeutics.     

Purification,characterization, sequencing and molecular cloning of a novel cysteine methyltransferase that regulates trehalose-6-phosphate synthase from Saccharomyces cerevisiae

Background

In Saccharomyces cerevisiae methylation at cysteine residue displayed enhanced activity of trehalose-6-phosphate synthase (TPS).

Methods

The cysteine methyltransferase (CMT) responsible for methylating TPS was purified and characterized. The amino acid sequence of the enzyme protein was determined by a combination of N-terminal sequencing and MALDI-TOF/TOF analysis. The nucleotide sequence of the CMT gene was determined, isolated from S. cerevisiae and expressed in E. coli. Targeted disruption of the CMT gene by PCR based homologous recombination in S. cerevisiae was followed by metabolite characterization in the mutant.

Results

The purified enzyme was observed to enhance the activity of TPS by a factor of 1.76. The 14 kDa enzyme was found to be cysteine specific. The optimum temperature and pH of enzyme activity was calculated as 30 °C and 7.0 respectively. The K_m V_max and K_cat against S-adenosyl-l-methionine (AdoMet) were 4.95 μM, 3.2 U/mg and 6.4 s^− 1 respectively. Competitive inhibitor S-Adenosyl-l-homocysteine achieved a K_i as 10.9 μM against AdoMet. The protein sequence contained three putative AdoMet binding motifs. The purified recombinant CMT activity exhibited similar physicochemical characteristics with the native counterpart. The mutant, Mataα, cmt:: kan^r exhibited almost 50% reduction in intracellular trehalose concentration.

Conclusion

A novel cysteine methyltransferase is purified, which is responsible for enhanced levels of trehalose in S. cerevisiae.

General significance

This is the first report about a cysteine methyltransferase which performs S methylation at cysteine residue regulating TPS activity by 50%, which resulted in an increase of the intercellular stress sugar, trehalose.     

Protein disulfide isomerase and glutathione are alternative substrates in the one Cys catalytic cycle of glutathione peroxidase 7

Background

Mammalian GPx7 is a monomeric glutathione peroxidase of the endoplasmic reticulum (ER), containing a Cys redox center (CysGPx). Although containing a peroxidatic Cys (C_P) it lacks the resolving Cys (C_R), that confers fast reactivity with thioredoxin (Trx) or related proteins to most other CysGPxs.

Methods

Reducing substrate specificity and mechanism were addressed by steady-state kinetic analysis of wild type or mutated mouse GPx7. The enzymes were heterologously expressed as a synuclein fusion to overcome limited expression. Phospholipid hydroperoxide was the oxidizing substrate. Enzyme–substrate and protein–protein interaction were analyzed by molecular docking and surface plasmon resonance analysis.

Results

Oxidation of the C_P is fast (k_+ 1 > 10³ M^− 1 s^− 1), however the rate of reduction by GSH is slow (k′_+ 2 = 12.6 M^− 1 s^− 1) even though molecular docking indicates a strong GSH–GPx7 interaction. Instead, the oxidized C_P can be reduced at a fast rate by human protein disulfide isomerase (HsPDI) (k_+ 1 > 10³ M^− 1 s^− 1), but not by Trx. By surface plasmon resonance analysis, a K_D = 5.2 μM was calculated for PDI–GPx7 complex. Participation of an alternative non-canonical C_R in the peroxidatic reaction was ruled out. Specific activity measurements in the presence of physiological reducing substrate concentration, suggest substrate competition in vivo.

Conclusions

GPx7 is an unusual CysGPx catalyzing the peroxidatic cycle by a one Cys mechanism in which GSH and PDI are alternative substrates.

General significance

In the ER, the emerging physiological role of GPx7 is oxidation of PDI, modulated by the amount of GSH.     

Entrapment of human flavin-containing monooxygenase 3 in the presence of gold nanoparticles: TEM,FTIR and electrocatalysis

Background

Nanosized particles of gold are widely used as advanced materials for enzyme catalysis investigations. In some bioanalytical methods these nanoparticles can be exploited to increase the sensitivity by enhancing electron transfer to the biological component i.e. redox enzymes such as drug metabolizing enzymes.

Methods

In this work, we describe the characterization of human flavin-containing monooxygenase 3 (hFMO3) in a nanoelectrode system based on AuNPs stabilized with didodecyldimethylammonium bromide (DDAB) on glassy carbon electrodes. Once confirmed by FTIR spectroscopy that in the presence of DDAB-AuNPs the structural integrity of hFMO3 is preserved, the influence of AuNPs on the electrochemistry of the enzyme was studied by cyclic voltammetry and square wave voltammetry.

Results

Our results show that AuNPs improve the electrochemical performance of hFMO3 on glassy carbon electrodes by enhancing the electron transfer rate and the current signal-to-noise ratio. Moreover, the electrocatalytic activity of hFMO3-DDAB-AuNP electrodes which was investigated in the presence of two well known substrates, benzydamine and sulindac sulfide, resulted in K_M values of 52 μM and 27 μM, with V_max of 8 nmol min^− 1 mg^− 1 and 4 nmol min^− 1 mg^− 1, respectively, which are in agreement with data obtained with the microsomal enzyme.

Conclusions

The immobilization of hFMO3 protein in DDAB stabilized AuNP electrodes improves the bioelectrochemical performance of this important phase I drug metabolizing enzyme.

General significance

This bio-analytical method can be considered as a promising advance in the development of new techniques suitable for the screening of novel hFMO3 metabolized pharmaceuticals.     

Development of peptide inhibitor as a therapeutic agent against head and neck squamous cell carcinoma (HNSCC) targeting p38α MAP kinase

Background

The p38α MAP kinase pathway is involved in inflammation, cell differentiation, growth, apoptosis and production of pro-inflammatory cytokines TNF-α and IL-1β. The overproduction of these cytokines plays an important role in cancer. The aim of this work was to design a peptide inhibitor on the basis of structural information of the active site of p38α.

Methods

A tetrapeptide, VWCS as p38α inhibitor was designed on the basis of structural information of the ATP binding site by molecular modeling. The inhibition study of peptide with p38α was performed by ELISA, binding study by Surface Plasmon Resonance and anti-proliferative assays by MTT and flow cytometry.

Results

The percentage inhibition of designed VWCS against pure p38α protein and serum of HNSCC patients was 70.30 and 71.5%, respectively. The biochemical assay demonstrated the K_D and IC₅₀ of the selective peptide as 7.22 × 10^− 9 M and 20.08 nM, respectively. The VWCS as inhibitor significantly reduced viability of oral cancer KB cell line with an IC₅₀ value of 10 μM and induced apoptosis by activating Caspase 3 and 7.

Conclusions

VWCS efficiently interacted at the ATP binding pocket of p38α with high potency and can be used as a potent inhibitor in case of HNSCC.

General significance

VWCS can act as an anticancer agent as it potentially inhibits the cell growth and induces apoptosis in oral cancer cell-line in a dose as well as time dependent manner. Hence, p38α MAP kinase inhibitor can be a potential therapeutic agent for human oral cancer.     

Whole blood,flow-chamber studies in real-time indicate a biphasic role for thymosin β-4 in platelet adhesion

Background

Thymosin beta 4 (Tβ₄) is a major actin sequestering peptide present in most mammalian cells. It also acts as an anti-inflammatory agent and promotes corneal wound healing.

Methods

In the present study, we constructed a four channel cylindrical flow chambers out of polydimethylsiloxane (PDMS) on microscope coverslips. The platelet-binding proteins–fibrinogen and collagen–were immobilized onto the middle ~ 25% of the inner cylindrical surface. The flow method introduced here was employed to determine the effect of Tβ₄, on the deposition of ADP-activated platelets onto fibrinogen cross-linked flow chambers.

Results

The binding data from the flow chambers indicated that the both the rate constant of platelet deposition (average: 0.026 ± 0.0015 s^− 1, corresponding to a half-life of 26.7 s) and the total number of deposited platelets were independent of the platelet binding protein and the activating agent. Our results show that low concentrations of Tβ₄ (0.2 μM to 0.5 μM) increased both the rate constant of platelet deposition by ~ 1.5-fold (i.e. half-life decreased from 26.7 s to 17.6 s) and the total number of deposited platelets by ~ 3-fold. However at higher concentrations (> 1 μM) the Tβ₄-potentiating effect was diminished to near control levels. Tβ₄ did interact with fibrinogen with an estimated K_D of ~ 126 ± 18 nM or 66 ± 20 nM under equilibrium or flow, respectively.

Conclusion

These results suggest that Tβ₄ could potentially increase the affinity of platelet receptors for their ligands thus promoting platelet deposition. Tβ₄ could also bind to fibrinogen and as its concentration increased would prevent platelet–fibrinogen interactions resulting in the attenuation of platelet deposition.

General significance

This work suggests that Tβ₄ might have a dual role in platelet function.     

Inhibition by polyamines of the hammerhead ribozyme from a Chrysanthemum chlorotic mottle viroid

Background

Viroids are the smallest pathogens known to date. They infect plants and cause considerable economic losses. The members of the Avsunviroidae family are known for their capability to form hammerhead ribozymes (HHR) that catalyze self-cleavage during their rolling circle replication.

Methods

In vitro inhibition assays, based on the self-cleavage kinetics of the hammerhead ribozyme from a Chrysanthemum chlorotic mottle viroid (CChMVd-HHR) were performed in the presence of various putative inhibitors.

Results

Aminated compounds appear to be inhibitors of the self-cleavage activity of the CChMVd HHR. Surprisingly the spermine, a known activator of the autocatalytic activity of another hammerhead ribozyme in the presence or absence of divalent cations, is a potent inhibitor of the CChMVd-HHR with K_i of 17 ± 5 μM. Ruthenium hexamine and TMPyP4 are also efficient inhibitors with K_i of 32 ± 5 μM and IC₅₀ of 177 ± 5 nM, respectively.

Conclusions

This study shows that polyamines are inhibitors of the CChMVd-HHR self-cleavage activity, with an efficiency that increases with the number of their amino groups.

General significance

This fundamental investigation is of interest in understanding the catalytic activity of HHR as it is now known that HHR are present in the three domains of life including in the human genome. In addition these results emphasize again the remarkable plasticity and adaptability of ribozymes, a property which might have played a role in the early developments of life and must be also of significance nowadays for the multiple functions played by non-coding RNAs.     

Endothelium-independent vasorelaxation effects of 16-O-acetyldihydroisosteviol on isolated rat thoracic aorta

Aims

The aim of this study is to investigate the vasorelaxant effect of 16-O-acetyldihydroisosteviol (ADIS) and its underlying mechanisms in isolated rat aorta.

Main methods

Rat aortic rings were isolated, suspended in organ baths containing Kreb's solution, maintained at 37 °C, and mounted on tungsten wire and continuously bubbled with a mixture of 95% O₂ and 5% CO₂ under a resting tension of 1 g. The vasorelaxant effects of ADIS were investigated by means of isometric tension recording experiment.

Key findings

ADIS (0.1 μM–3 mM) induced relaxation of aortic rings pre-contracted by phenylephrine (PE, 10 μM) and KCl (80 mM) with intact-endothelium (E_max = 79.26 ± 3.74 and 79.88 ± 3.79, respectively) or denuded-endothelium (E_max = 88.05 ± 3.69 and 78.22 ± 6.86, respectively). In depolarization Ca²⁺-free solution, ADIS inhibits calcium chloride (CaCl₂)-induced contraction in endothelium-denuded rings in a concentration-dependent manner. In addition, ADIS attenuates transient contractions in Ca²⁺-free medium containing EGTA (1 mM) induced by PE (10 μM) and caffeine (20 mM). By contrast, relaxation was not affected by tetraethylammonium (TEA, 5 mM), 4-aminopyridine (4-AP, 1 mM), glibenclamide (10 μM), barium chloride (BaCl₂, 1 mM), and 1H-[1,2,3]oxadiazolo[4,3-α]quinoxalin-1-one (ODQ, 1 μM).

Significance

These findings reveal the vasorelaxant effect of ADIS, through endothelium-independent pathway. It acts as a Ca^2 + channel blocker through both intracellular and extracellular Ca^2 + release.     

Selective cytotoxicity of intense nanosecond-duration electric pulses in mammalian cells

Background

Nanosecond electric pulses (EP) disrupt cell membrane and organelles and cause cell death in a manner different from the conventional irreversible electroporation. We explored the cytotoxic effect of 10-ns EP (quantitation, mechanisms, efficiency, and specificity) in comparison with 300-ns, 1.8- and 9-μs EP.

Methods

Effects in Jurkat and U937 cells were characterized by survival assays, DNA electrophoresis and flow cytometry.

Results

10-ns EP caused apoptotic or necrotic death within 2–20 h. Survival (S, %) followed the absorbed dose (D, J/g) as: S = αD^(−K), where coefficients K and α determined the slope and the “shoulder” of the survival curve. K was similar in all groups, whereas α was cell type- and pulse duration-dependent. Long pulses caused immediate propidium uptake and phosphatidylserine (PS) externalization, whereas 10-ns pulses caused PS externalization only.

Conclusions

1.8- and 9-μs EP cause cell death efficiently and indiscriminately (LD₅₀ 1–3 J/g in both cell lines); 10-ns EP are less efficient, but very selective (LD₅₀ 50–80 J/g for Jurkat and 400–500 J/g for U937); 300-ns EP show intermediate effects. Shorter EP open propidium-impermeable, small membrane pores (”nanopores”), triggering different cell death mechanisms.

General significance

Nanosecond EP can selectively target certain cells in medical applications like tumor ablation.     

Investigation of roles of divalent cations in Shewanella oneidensis pellicle formation reveals unique impacts of insoluble iron

Background

Bacteria adopt a variety of lifestyles in their natural habitats and can alternate among different lifestyles in response to environmental changes. At high cell densities, bacteria can form extracellular matrix encased cell population on submerged tangible surfaces (biofilms), or at the air–liquid interface (pellicles). Compared to biofilm, pellicle lifestyle allows for better oxygen access, but is metabolically more costly to maintain. Further understanding of pellicle formation and environmental cues that influence cellular choices between these lifestyles will definitely improve our appreciation of bacterial interaction with their environments.

Methods

Shewanella oneidensis cells were cultured in 24-well plates with supplementation of varied divalent cations, and pellicles formed under such conditions were evaluated. Mutants defective in respiration of divalent cations were used to further characterize and confirm unique impacts of iron.

Results and conclusions

Small amount of Fe^2 + was essential for pellicle formation, but presence of over-abundant iron (0.3 mM Fe^2 + or Fe^3 +) led to pellicle disassociation without impairing growth. Such impacts were found due to S. oneidensis-mediated formation of insoluble alternative electron acceptors (i.e., Fe₃O₄) under physiologically relevant conditions. Furthermore, we demonstrated that cells preferred a lifestyle of forming biofilm and respiring on such insoluble electron acceptors under tested conditions, even to living in pellicles.

General significance

Our finding suggests that bacterial lifestyle choice involves balanced evaluation of multiple aspects of environmental conditions, and yet-to-be-characterized signaling mechanism is very likely underlying such processes.     

Comparison of quantum dot-binding protein tags: Affinity determination by ultracentrifugation and FRET

Background

Hybrid complexes of proteins and colloidal semiconductor nanocrystals (quantum dots, QDs) are of increasing interest in various fields of biochemistry and biomedicine, for instance for biolabeling or drug transport. The usefulness of protein–QD complexes for such applications is dependent on the binding specificity and strength of the components. Often the binding properties of these components are difficult and time consuming to assess.

Methods

In this work we characterized the interaction between recombinant light harvesting chlorophyll a/b complex (LHCII) and CdTe/CdSe/ZnS QDs by using ultracentrifugation and fluorescence resonance energy transfer (FRET) assay experiments. Ultracentrifugation was employed as a fast method to compare the binding strength between different protein tags and the QDs. Furthermore the LHCII:QD stoichiometry was determined by separating the protein–QD hybrid complexes from unbound LHCII via ultracentrifugation through a sucrose cushion.

Results

One trimeric LHCII was found to be bound per QD. Binding constants were evaluated by FRET assays of protein derivatives carrying different affinity tags. A new tetra-cysteine motif interacted more strongly (K_a = 4.9 ± 1.9 nM^− 1) with the nanoparticles as compared to a hexahistidine tag (His₆ tag) (K_a ~ 1 nM^− 1).

Conclusion

Relative binding affinities and binding stoichiometries of hybrid complexes from LHCII and quantum dots were identified via fast ultracentrifugation, and binding constants were determined via FRET assays.

General significance

The combination of rapid centrifugation and fluorescence-based titration will be useful to assess the binding strength between different types of nanoparticles and a broad range of proteins.     

Expression of the hypoxia-inducible monocarboxylate transporter MCT4 is increased in triple negative breast cancer and correlates independently with clinical outcome

Background

¹⁸Fluor-deoxy-glucose PET-scanning of glycolytic metabolism is being used for staging in many tumors however its impact on prognosis has never been studied in breast cancer.

Methods

Glycolytic and hypoxic markers: glucose transporter (GLUT1), carbonic anhydrase IX (CAIX), monocarboxylate transporter 1 and 4 (MCT1, 4), MCT accessory protein basigin and lactate-dehydrogenase A (LDH-A) were assessed by immunohistochemistry in two cohorts of breast cancer comprising 643 node-negative and 127 triple negative breast cancers (TNBC) respectively.

Results

In the 643 node-negative breast tumor cohort with a median follow-up of 124 months, TNBC were the most glycolytic (≈70%), followed by Her-2 (≈50%) and RH-positive cancers (≈30%). Tumoral MCT4 staining (without stromal staining) was a strong independent prognostic factor for metastasis-free survival (HR = 0.47, P = 0.02) and overall-survival (HR = 0.38, P = 0.002). These results were confirmed in the independent cohort of 127 cancer patients.

Conclusion

Glycolytic markers are expressed in all breast tumors with highest expression occurring in TNBC. MCT4, the hypoxia-inducible lactate/H⁺ symporter demonstrated the strongest deleterious impact on survival. We propose that MCT4 serves as a new prognostic factor in node-negative breast cancer and can perhaps act soon as a theranostic factor considering the current pharmacological development of MCT4 inhibitors.     

Histological and functional renal alterations caused by Bothrops alternatus snake venom: Expression and activity of Na/K-ATPase

Background

Acute renal failure is a serious complication of human envenoming by Bothrops snakes. The ion pump Na⁺/K⁺-ATPase has an important role in renal tubule function, where it modulates sodium reabsorption and homeostasis of the extracellular compartment. Here, we investigated the morphological and functional renal alterations and changes in Na⁺/K⁺-ATPase expression and activity in rats injected with Bothrops alternatus snake venom.

Methods

Male Wistar rats were injected with venom (0.8 mg/kg, i.v.) and renal function was assessed 6, 24, 48 and 72 h and 7 days post-venom. The rats were then killed and renal Na⁺/K⁺-ATPase activity was assayed based on phosphate release from ATP; gene and protein expressions were assessed by real time PCR and immunofluorescence microscopy, respectively.

Results

Venom caused lobulation of the capillary tufts, dilation of Bowman's capsular space, F-actin disruption in Bowman's capsule and renal tubule brush border, and deposition of collagen around glomeruli and proximal tubules that persisted seven days after envenoming. Enhanced sodium and potassium excretion, reduced proximal sodium reabsorption, and proteinuria were observed 6 h post-venom, followed by a transient decrease in the glomerular filtration rate. Gene and protein expressions of the Na⁺/K⁺-ATPase α₁ subunit were increased 6 h post-venom, whereas Na⁺/K⁺-ATPase activity increased 6 h and 24 h post-venom.

Conclusions

Bothrops alternatus venom caused marked morphological and functional renal alterations with enhanced Na⁺/K⁺-ATPase expression and activity in the early phase of renal damage.

General significance

Enhanced Na⁺/K⁺-ATPase activity in the early hours after envenoming may attenuate the renal dysfunction associated with venom-induced damage.     

Pivotal role of cAMP in the activation of liver glycogen breakdown in high-fat diet fed mice

Aims

Liver glycogen catabolism was evaluated in male Swiss mice fed a high-fat diet rich in saturated fatty acids (HFD) or normal fat diet (NFD) during one week.

Main methods

Liver glycogenolysis (LG) and liver glucose production (LGP) were measured either under basal or stimulated conditions (infusion of glycogenolytic agents). Thus, isolated perfused livers from HFD and NFD mice were infused with glycogenolytic agents, i.e., glucagon, epinephrine, phenylephrine, isoproterenol, adenosine-3′-5′-cyclic monophosphate (cAMP), N⁶,2′-O-dibutyryl-cAMP (DB-cAMP), 8-bromoadenosine-cAMP (8-Br-cAMP) or N⁶-monobutyryl-cAMP (N6-MB-cAMP). Moreover, glycemia and liver glycogen content were measured.

Key findings

Glycemia, liver glycogen content and basal rate of LGP and LG were not influenced by the HFD. However, LGP and LG were lower (p < 0.05) in HFD mice during the infusions of glucagon (1 nM), epinephrine (20 μM) or phenylephrine (20 μM). In contrast, the activation of LGP and LG during the infusion of isoproterenol (20 μM) was not different (HFD vs. NFD). Because glucagon showed the most prominent response, the effect of cAMP, its intracellular mediator, on LGP and LG was investigated. cAMP (150 μM) showed lower activation of LGP and LG in the HFD group. However, the activation of LGP and LG was not influenced by HFD whether DB-cAMP (3 μM), 8-Br-cAMP (3 μM) or N6-MB-cAMP (3 μM) were used.

Significance

The activation of LGP and LG depends on the intracellular availability of cAMP. It can be concluded that cAMP played a pivotal role on the activation of LG in high-fat diet fed mice.     

In vivo enzymatic activity of acetylCoA synthetase in skeletal muscle revealed by C turnover from hyperpolarized [1-C]acetate to [1-C]acetylcarnitine

Background

Acetate metabolism in skeletal muscle is regulated by acetylCoA synthetase (ACS). The main function of ACS is to provide cells with acetylCoA, a key molecule for numerous metabolic pathways including fatty acid and cholesterol synthesis and the Krebs cycle.

Methods

Hyperpolarized [1-¹³C]acetate prepared via dissolution dynamic nuclear polarization was injected intravenously at different concentrations into rats. The ¹³C magnetic resonance signals of [1-¹³C]acetate and [1-¹³C]acetylcarnitine were recorded in vivo for 1 min. The kinetic rate constants related to the transformation of acetate into acetylcarnitine were deduced from the 3 s time resolution measurements using two approaches, either mathematical modeling or relative metabolite ratios.

Results

Although separated by two biochemical transformations, a kinetic analysis of the ¹³C label flow from [1-¹³C]acetate to [1-¹³C]acetylcarnitine led to a unique determination of the activity of ACS. The in vivo Michaelis constants for ACS were K_M = 0.35 ± 0.13 mM and V_max = 0.199 ± 0.031 μmol/g/min.

Conclusions

The conversion rates from hyperpolarized acetate into acetylcarnitine were quantified in vivo and, although separated by two enzymatic reactions, these rates uniquely defined the activity of ACS. The conversion rates associated with ACS were obtained using two analytical approaches, both methods yielding similar results.

General significance

This study demonstrates the feasibility of directly measuring ACS activity in vivo and, since the activity of ACS can be affected by various pathological states such as cancer or diabetes, the proposed method could be used to non-invasively probe metabolic signatures of ACS in diseased tissue.     

Effects of sub-chronic aluminum chloride exposure on rat ovaries

Aims

This experiment investigated the effects of sub-chronic aluminum chloride (AlCl₃) exposure on rat ovaries.

Main methods

Eighty female Wistar (5 weeks old) rats, weighed 110–120 g, were randomly divided into four treatment groups: control group (CG), low-dose group (LG, 64 mg/kg BW AlCl₃), mid-dose group (MG, 128 mg/kg BW AlCl₃) and high-dose group (HG, 256 mg/kg BW AlCl₃). The AlCl₃ was administered in drinking water for 120 days. The ovarian ultrastructure was observed. The activities of acid phosphatase (ACP), alkaline phosphatase (ALP), succinate dehydrogenase (SDH), Na⁺–K⁺-ATPase, Mg^2 +-ATPase and Ca^2 +-ATPase, the contents of Fe, Cu and Zn, and the protein expression of follicle-stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHR) in the ovary were determined.

Key findings

The results showed that the structure of the ovary was disrupted, the activities of ALP, ACP, SDH, Na⁺–K⁺-ATPase, Mg^2 +-ATPase and Ca^2 +-ATPase, the contents of Zn, Fe and the protein expression of FSHR and LHR were lowered, and the content of Cu was increased in AlCl₃-treated rats than those in control.

Significance

The results indicate that sub-chronic AlCl₃ exposure caused the damage of the ovarian structure, the disturbed metabolism of Fe, Zn and Cu and the decreased activities of Na⁺–K⁺-ATPase, Mg^2 +-ATPase and Ca^2 +-ATPase in the ovary, which could result in suppressed energy supply in the ovary. A combination of suppression of energy supply and reduction of expression of FSHR and LHR could inhibit ovulation and corpus luteum development, leading to infertility in female rats.     

Kinetic characterization of recombinant mouse retinal dehydrogenase types 3 and 4 for retinal substrates

Background

Retinal dehydrogenases (RALDHs) catalyze the dehydrogenation of retinal into retinoic acids (RAs), which are required for embryogenesis and tissue differentiation. This study sought to determine the detailed kinetic properties of 2 mouse RALDHs, namely RALDH3 and 4, for retinal isomer substrates, to better define their specificities in RA isomer synthesis.

Methods

RALDH3 and 4 were expressed in Escherichia coli as His-tagged proteins and affinity-purified. Enzyme kinetics were performed with retinal isomer substrates. The enzymatic products were analyzed by high pressure liquid chromatography.

Results

RALDH3 oxidized all-trans retinal with high catalytic efficiency (V_max/K_m = 77.9) but did not show activity for either 9-cis or 13-cis retinal substrates. On the other hand, RALDH4 was inactive for all-trans retinal substrate, exhibited high activity for 9-cis retinal oxidation (V_max/K_m = 27.4), and oxidized 13-cis retinal with lower catalytic efficiency (V_max/K_m = 8.24). β-ionone, a potent inhibitor of RALDH4 activity, suppressed 9-cis and 13-cis retinal oxidation competitively with inhibition constants of 0.60 and 0.32, respectively, but had no effect on RALDH3 activity. The divalent cation MgCl₂ activated 13-cis retinal oxidation by RALDH4 by 3-fold, did not significantly influence 9-cis retinal oxidation, and slightly activated RALDH3 activity.

Conclusions

These data extend the kinetic characterization of RALDH3 and 4, providing their specificities for retinal isomer substrates.

General significance

The kinetic characterization of RALDHs should give useful information in determining amino acid residues that are involved in the specificity for retinal isomers and on the role of these enzymes in the synthesis of RAs in specific tissues.